Identification of structural and functional O-GlcNAc-bearing proteins in Xenopus laevis oocyte.
Résumé
O-GlcNAcylation (or O-GlcNAc) is an abundant and reversible glycosylation type found within the cytosolic and the nuclear compartments. We have previously described the sudden O-GlcNAcylation increase occurring during the Xenopus laevis oocyte G2/M transition and we have demonstrated that the inhibition of the O-GlcNAc transferase (OGT) blocked this process, showing that the O-GlcNAcylation dynamism interferes with the cell cycle progression. In this work, we have identified proteins that are O-GlcNAc modified during the G2/M transition. Due to a low expression of O-GlcNAcylation in Xenopus oocyte, classical enrichment of O-GlcNAc-bearing proteins, using O-GlcNAc-directed antibodies or Wheat Germ Agglutinin-lectin affinity, were hard to apply albeit these techniques allowed the identification of actin and Erk2. Therefore, another strategy based on an in vitro enzymatic labeling of O-GlcNAc residues with GalNAz (i.e. azido GalNAc), followed by a chemical addition of a biotin-alkyne probe and by enrichment of the tagged-proteins on avidin-beads was used. Bound-proteins were analysed by nanoLC-nanoESI-MS/MS allowing for the identification of an average of twenty Xenopus laevis oocyte O-GlcNAcylated proteins. In addition to actin and beta-tubulin, we identified metabolic/functional proteins such as PP2A, PCNA, TER ATPase, aldolase, lactate dehydrogenase and ribosomal proteins. This labelling allowed for the mapping of a major O-GlcNAcylation site within the 318-324 region of beta-actin. Furthermore, immunofluorescence microscopy enabled the direct visualization of O-GlcNAcylation and OGT on the meiotic spindle as well as the observation that chromosomal-bound proteins were enriched in O-GlcNAc and OGT. The biological relevance of this post-translational modification (PTM) both on microtubules and on chromosomes remains to be determined. However, the mapping of the O-GlcNAcylation sites will help to underline the function of this PTM on each identified protein and will provide a better understanding of O-GlcNAcylation in the control of the cell cycle.